Elevated osteoclast (OC) formation and/or activity are implicated in the pathogenesis of various bone disorders. Current antiresorptive (anti-OC formation and/or function) drugs either lack satisfactory efficacy or cause serious side effects. OC formation requires two key factors: the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-?B ligand (RANKL). RANKL regulates OC formation and function by activating its receptor RANK (the receptor activator of NF-?B) on OC precursors and mature OCs. RANK, a member of the tumor necrosis factor receptor (TNFR) family, transduces intracellular signals by recruiting adaptor proteins including TNFR associated factors (TRAFs) through specific motifs in the cytoplasmic domain. The unraveling of the RANKL/RANK system has not only greatly advanced our understanding of OC biology but also provided an opportunity to develop more effective antiresorptive drugs. The current effort has primarily been focused on developing therapeutics blocking RANK/RANKL interactions, which are likely to have side effects due to the involvement of the system in multiple biological processes. Recently we identified three functional TRAF-binding motifs in the RANK cytoplasmic domain (Motif 1:PFQEP369-373; Motif 2: PVQEET559-564 & Motif 3: PVQEQG604-609). These motifs regulate OC formation and function by utilizing different TRAF proteins to form distinct signaling complexes. However, while it has been established that Motif 1 recruits TRAF6 to form a multiple protein signaling complex to activate numerous signaling pathways, the precise signaling mechanism by which Motif 2 and Motif 3 activate downstream signals have not been fully understood. Moreover, these motifs regulate OC formation and function to different extent. While Motif 2 and Motif 3 are very potent in mediating OC formation and function, Motif 1 plays a minimal role in these processes, instead being the primary actor in RANKL-mediated immune function. Thus, Motifs 2 and 3 can serve as potent and specific antiresorptive targets. In this application, we propose to develop and configure novel cell-based assay systems for high throughput screen (HTS) to identify compounds blocking signaling from Motif 2 and 3. To this end, we seek to achieve the following two specific aims: Aim 1: Develop highly efficient and robotic cell-based assays for identifying compounds blocking the signaling pathways activated by Motif 2 and Motif 3; Aim 2: Configure the cell-based assays for HTS; Aim 3: Submit the cell based assays to the Molecular Libraries Screening Center Network (MLSCN) to identify compounds inhibiting Motif 2- and Motif-3 mediated signaling. Significantly, the compounds can not only serve as useful molecular probes for elucidating the signaling mechanism of Motif 2-/Motif 3 but may also be further developed as therapeutics for osteoporosis, bone loss in rheumatoid arthritis (RA) and tumor bone metastasis. [unreadable] [unreadable] [unreadable]